Factor ix gene therapy

ABSTRACT

The invention relates to a new, more potent, coagulation Factor IX (FIX) expression cassette for gene therapy of Haemophilia B (HB). Disclosed is a vector for expressing factor IX protein, the vector comprising a promoter, a nucleotide sequence encoding for a functional factor IX protein, and an intron sequence, wherein the intron sequence is positioned between exon 1 and exon 2 of the nucleotide sequence encoding for a functional factor IX protein, and wherein the intron sequence has at least 80% identity to the sequence of SEQ ID NO. 1 as disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/563,193, filed on Sep. 6, 2019, which is a continuation of U.S.patent application Ser. No. 15/525,836, filed May 10, 2017, which is anational phase entry under 35 U.S.C. § 371 of International PatentApplication PCT/GB2015/053438, filed Nov. 12, 2015, designating theUnited States of America and published in English as InternationalPatent Publication WO/2016/075473 on May 19, 2016, which claims thebenefit under Article 8 of the Patent Cooperation Treaty to UnitedKingdom Patent Application Serial No. 1420139.6, filed Nov. 12, 2014,the entirety of each of which are incorporated herein by this reference.

TECHNICAL FIELD

The invention relates to a new more potent coagulation factor IX (FIX)expression cassette for gene therapy of haemophilia B (HB).

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(C) OR (E)—SEQUENCE LISTINGSUBMITTED AS A TXT FILE

Pursuant to 37 C.F.R. § 1.821(c) or (e), files containing a TXT versionof the Sequence Listing have been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

BACKGROUND

Haemophilia B, an X-linked life threatening bleeding disorder affects1:30,000 males. Current treatment involves frequent intravenousinjections (2-3 times per week) of FIX protein. This treatment is highlyeffective at arresting bleeding but it is not curative and is extremelyexpensive (£150,000/patient/year), thus making it unaffordable by themajority of haemophilia B patients in the World. Gene therapy for HBoffers the potential for a cure through persistent, endogenousproduction of FIX following the transfer of a normal copy of the FIXgene to an affected patient. Even a small rise in circulating FIX to >1%of normal levels can significantly ameliorate the severe bleedingphenotype.

The inventors have recently successfully piloted a gene therapy approachusing adeno-associated viral vectors (AAV) to mediate transfer andexpression of the gene for normal coagulation factor IX (FIX) in theliver. Preliminary results of this successful academic, investigatorled, trial were published in the New England Journal of Medicine, withexperts in the field lauding the work, and calling it a “landmarkstudy”.

BRIEF SUMMARY

Despite the initial success, some obstacles remain to the overridinggoal of making AAV-mediated transfer of the normal FIX gene theworld-wide curative standard-of-care. Foremost among these is the body'simmune response to cells that have been transduced with the viral vectorthat resulted in asymptomatic, transient elevation of serum liverenzymes, suggesting local inflammation in the liver. Treatment with ashort course of steroids led to rapid normalisation of the liver enzymesand continued expression of FIX, but at levels below those that had beengenerated before the episodes of liver inflammation. Since thisobservation only occurred at the high dose level, current efforts arefocused on improving the potency and transduction efficiency of AAVvectors so that therapeutic gene transfer can be achieved with lower,potentially safer vector doses. To this end, the inventors havedeveloped a new AAV expression cassette, referred to asHLP2-TI-codop-FIX, which mediates transgene expression at levels thatare up to four fold higher than previously observed with theself-complementary (sc) LP1-hFIXco cassette used in the previousclinical trial. HLP2-TI-codop-FIX improves the safety profile of AAVmediated gene transfer whilst reducing the burden on vector production.

The aspects of HLP2-TI-codop-FIX which make it different from previouslyused vectors include the following:

1. It is a single stranded vector. This allows a larger transgene to bepackaged in the AAV than with self-complementary AAV;

2. A new synthetic liver promoter (HLP2). This promoter is a modifiedpromoter which contains an extra enhancer region to increase expression;

3. Truncated 299 bp intron (TI) derived by engineering sequences fromwild type FIX intron 1A which spans 6.2 kb. The truncated intron (TI) inthis expression cassette is placed in its native position between exon 1and exon 2 of the FIX. Despite its smaller size, it increases expressionlevels by 1.6 fold over that observed with wild type full length intron1A; and

4. New codon optimised FIX to increase expression over wild-typesequence by almost two fold (FIG. 2).

In a first aspect of the invention, there is provided a vector forexpressing factor IX protein, the vector comprising a promoter, anucleotide sequence encoding for a functional factor IX protein and anintron sequence, wherein the intron sequence is positioned between exon1 and exon 2 of the nucleotide sequence encoding for a functional factorIX protein, and wherein the intron sequence has at least 80% identity tothe sequence of SEQ ID NO. 1.

The intron sequence in the vector is derived from intron 1A of the wildtype FIX gene. It has been found that truncating the sequence of intron1A causes expression of the vector to be increased. It is thought thatthe truncation of intron 1A may delete a repressor element in theintron.

Truncation of the intron 1A sequence also results in the nucleotidesequence of the vector being shorter which allows more efficientpackaging of the vector in a viral delivery system.

In the vector of the invention, the intron sequence is positionedbetween exon 1 and exon 2 of the nucleotide sequence encoding for afunctional factor IX protein. The complete sequence of the human FIXgene is well known, including the sequences of the introns and exons.For example, this information can be found on Genbank(http://www.ncbi.nlm.nih.gov/genbank) under accession numbers J00137.1;B C109215. 1; BC109214.1; AB186358.1; and FR846239.1. Therefore, it iswell within the abilities of a skilled person to determine where in theFIX coding sequence the intron sequence is located and in particular,between exon 1 and 2 of the FIX gene. Generally, when protein codingsequences are incorporated into vectors, the coding sequence does notcontain any introns.

The intron sequence has at least 80% identity to the sequence of SEQ IDNO. 1. In some embodiments, the intron sequence has at least 82%identity to the sequence of SEQ ID NO. 1. In other embodiments, theintron sequence has at least 84% identity to the sequence of SEQ IDNO. 1. In particular embodiments, the intron sequence has at least 86%identity to the sequence of SEQ ID NO. 1. In further embodiments, theintron sequence has at least 88% identity to the sequence of SEQ IDNO. 1. In some embodiments, the intron sequence has at least 90%identity to the sequence of SEQ ID NO. 1. In other embodiments, theintron sequence has at least 91% identity to the sequence of SEQ IDNO. 1. In particular embodiments, the intron sequence has at least 92%identity to the sequence of SEQ ID NO. 1. In further embodiments, theintron sequence has at least 93% identity to the sequence of SEQ IDNO. 1. In some embodiments, the intron sequence has at least 94%identity to the sequence of SEQ ID NO. 1. In other embodiments, theintron sequence has at least 95% identity to the sequence of SEQ IDNO. 1. In particular embodiments, the intron sequence has at least 96%identity to the sequence of SEQ ID NO. 1. In further embodiments, theintron sequence has at least 97% identity to the sequence of SEQ IDNO. 1. In some embodiments, the intron sequence has at least 98%identity to the sequence of SEQ ID NO. 1. In other embodiments, theintron sequence has at least 99% identity to the sequence of SEQ IDNO. 1. In particular embodiments, the intron sequence has the sequenceof SEQ ID NO. 1.

The truncated intron sequence is preferably between 270 and 330nucleotides in length. In some embodiments, the intron sequence isbetween 280 and 320 nucleotides in length. In other embodiments, theintron sequence is between 290 and 310 nucleotides in length. Inparticular embodiments, the intron sequence is between 295 and 305nucleotides in length. In specific embodiments, the intron sequence isbetween 295 and 300 nucleotides in length.

The vector contains a nucleotide sequence encoding for a functionalfactor IX protein so that when this sequence is expressed, a functionalFIX protein is produced by the cell in which the vector is contained.When expressed in a subject, e.g. a human patient, the functional FIXprotein is one which can take part in the coagulation cascade to allowblood dotting to take place. This causes a decrease in the time it takesfor blood to clot in a subject suffering from haemophilia B. Thefunctional FIX protein can be activated to produce the enzymaticallyactive factor IXa which can convert factor X to factor Xa.

The sequence of the FIX protein produced by expression of the vector maybe the wild type FIX sequence. In one embodiment, the nucleotidesequence encoding for a functional factor IX protein consists of exons 1to 5 of the FIX gene. The FIX gene normally contains 8 exons, with exons6 to 8 encoding an untranslated region. The advantage of using a shortersequence is that it can be incorporated into a vector more easily andmore effectively.

As mentioned above, the sequence of the FIX gene is well known to askilled person and therefore, it would be well within the capabilitiesof a skilled person to produce a nucleotide sequence encoding for afunctional factor IX protein.

The nucleotide sequence encoding for a functional FIX protein preferablyhas at least 80% identity to the sequence of SEQ ID NO. 2. In someembodiments, the nucleotide sequence has at least 82% identity to thesequence of SEQ ID NO. 2. In other embodiments, the nucleotide sequencehas at least 84% identity to the sequence of SEQ ID NO. 2. In furtherembodiments, the nucleotide sequence has at least 86% identity to thesequence of SEQ ID NO. 2. In particular embodiments, the nucleotidesequence has at least 88% identity to the sequence of SEQ ID NO. 2. Insome embodiments, the nucleotide sequence has at least 90% identity tothe sequence of SEQ ID NO. 2. In other embodiments, the nucleotidesequence has at least 91% identity to the sequence of SEQ ID NO. 2. Infurther embodiments, the nucleotide sequence has at least 92% identityto the sequence of SEQ ID NO. 2. In particular embodiments, thenucleotide sequence has at least 93% identity to the sequence of SEQ IDNO. 2. In some embodiments, the nucleotide sequence has at least 94%identity to the sequence of SEQ ID NO. 2. In other embodiments, thenucleotide sequence has at least 95% identity to the sequence of SEQ IDNO. 2. In further embodiments, the nucleotide sequence has at least 96%identity to the sequence of SEQ ID NO. 2. In particular embodiments, thenucleotide sequence has at least 97% identity to the sequence of SEQ IDNO. 2. In some embodiments, the nucleotide sequence has at least 98%identity to the sequence of SEQ ID NO. 2. In other embodiments, thenucleotide sequence has at least 99% identity to the sequence of SEQ IDNO. 2. In preferred embodiments, the nucleotide sequence has thesequence of SEQ ID NO. 2.

When the nucleotide sequence encoding for a functional FIX protein hassequence identity to the sequence of SEQ ID NO. 2, this does not includethe intron sequence which is positioned between exon 1 and exon 2 of thenucleotide sequence encoding for a functional factor IX protein. Forexample, when the nucleotide sequence encoding for a functional FIXprotein has the sequence of SEQ ID NO. 2 and the intron sequence has thesequence of SEQ ID NO. 1, in the nucleotide sequence of the actualvector, the sequence of SEQ ID NO. 1 will appear within SEQ ID NO. 2.This means that there will be a portion of SEQ ID NO. 2 followed by SEQID NO. 1 followed by the remaining portion of SEQ ID NO. 2.

The sequence of SEQ ID NO. 2 is a codon optimised FIX nucleotidesequence in which the sequence of exons 3 to 5 has been codon optimised.The sequence of exons 1 and 2 of SEQ ID NO. 2 is the wild type FIXsequence. The sequence of exons 3 to 5 has not been codon optimised in anormal way. Instead, the codons have been selected based on the codonsused for proteins which are expressed at a high level in the liver. Thereason for this is that the vector is normally expressed in the liver.This special codon optimisation process has been found to produce anucleotide sequence which gives surprisingly high expression. The wildtype sequence has been used for exons 1 and 2 to help to ensure thatsplicing is not affected when the intron is removed during processing ofthe RNA molecule expressed from the nucleotide sequence.

As described above, the vector comprises a nucleotide sequence encodingfor a functional factor IX protein and an intron sequence, wherein theintron sequence is positioned between exon 1 and exon 2 of thenucleotide sequence encoding for a functional factor IX protein, andwherein the intron sequence has at least 80% identity to the sequence ofSEQ ID NO. 1. In various embodiments, exons 3 to 5 of the nucleotidesequence encoding for a functional factor IX protein has 80% identity tothe sequence of SEQ ID NO. 6. In some embodiments, the nucleotidesequence of exons 3 to 5 has at least 82% identity to the sequence ofSEQ ID NO. 6. In other embodiments, the nucleotide sequence of exons 3to 5 has at least 84% identity to the sequence of SEQ ID NO. 6. Infurther embodiments, the nucleotide sequence of exons 3 to 5 has atleast 86% identity to the sequence of SEQ ID NO. 6. In particularembodiments, the nucleotide sequence of exons 3 to 5 has at least 88%identity to the sequence of SEQ ID NO. 6. In some embodiments, thenucleotide sequence of exons 3 to 5 has at least 90% identity to thesequence of SEQ ID NO. 6. In other embodiments, the nucleotide sequenceof exons 3 to 5 has at least 91% identity to the sequence of SEQ ID NO.6. In further embodiments, the nucleotide sequence of exons 3 to 5 hasat least 92% identity to the sequence of SEQ ID NO. 6. In particularembodiments, the nucleotide sequence of exons 3 to 5 has at least 93%identity to the sequence of SEQ ID NO. 6. In some embodiments, thenucleotide sequence of exons 3 to 5 has at least 94% identity to thesequence of SEQ ID NO. 6. In other embodiments, the nucleotide sequenceof exons 3 to 5 has at least 95% identity to the sequence of SEQ ID NO.6. In further embodiments, the nucleotide sequence of exons 3 to 5 hasat least 96% identity to the sequence of SEQ ID NO. 6. In particularembodiments, the nucleotide sequence of exons 3 to 5 has at least 97%identity to the sequence of SEQ ID NO. 6. In some embodiments, thenucleotide sequence of exons 3 to 5 has at least 98% identity to thesequence of SEQ ID NO. 6. In other embodiments, the nucleotide sequenceof exons 3 to 5 has at least 99% identity to the sequence of SEQ ID NO.6. In preferred embodiments, the nucleotide sequence of exons 3 to 5 hasthe sequence of SEQ ID NO. 6.

The nucleotide sequence encoding for a functional FIX protein ispreferably between 1335 and 1435 nucleotides in length. In someembodiments, the nucleotide sequence encoding for a functional FIXprotein is between 1360 and 1410 nucleotides in length. In otherembodiments, the nucleotide sequence encoding for a functional FIXprotein is between 1375 and 1395 nucleotides in length. In particularembodiments, the nucleotide sequence encoding for a functional FIXprotein is between 1380 and 1390 nucleotides in length.

In some embodiments, the nucleotide sequence encoding for a functionalFIX protein, including the intron sequence between exons 1 and 2, has80% identity to the sequence of SEQ ID NO. 3. In some embodiments, thenucleotide sequence has at least 82% identity to the sequence of SEQ IDNO. 3. In other embodiments, the nucleotide sequence has at least 84%identity to the sequence of SEQ ID NO. 3. In further embodiments, thenucleotide sequence has at least 86% identity to the sequence of SEQ IDNO. 3. In particular embodiments, the nucleotide sequence has at least88% identity to the sequence of SEQ ID NO. 3. In some embodiments, thenucleotide sequence has at least 90% identity to the sequence of SEQ IDNO. 3. In other embodiments, the nucleotide sequence has at least 91%identity to the sequence of SEQ ID NO. 3. In further embodiments, thenucleotide sequence has at least 92% identity to the sequence of SEQ IDNO. 3. In particular embodiments, the nucleotide sequence has at least93% identity to the sequence of SEQ ID NO. 3. In some embodiments, thenucleotide sequence has at least 94% identity to the sequence of SEQ IDNO. 3. In other embodiments, the nucleotide sequence has at least 95%identity to the sequence of SEQ ID NO. 3. In further embodiments, thenucleotide sequence has at least 96% identity to the sequence of SEQ IDNO. 3. In particular embodiments, the nucleotide sequence has at least97% identity to the sequence of SEQ ID NO. 3. In some embodiments, thenucleotide sequence has at least 98% identity to the sequence of SEQ IDNO. 3. In other embodiments, the nucleotide sequence has at least 99%identity to the sequence of SEQ ID NO. 3. In preferred embodiments, thenucleotide sequence encoding for a functional FIX protein, including theintron sequence between exons 1 and 2, has the sequence of SEQ ID NO. 3.

Therefore, in preferred embodiments, the present invention provides avector for expressing factor IX protein, the vector comprising apromoter, and a nucleotide sequence encoding for a functional factor IXprotein, wherein an intron sequence is positioned between exon 1 andexon 2 of the nucleotide sequence encoding for a functional factor IXprotein, and wherein the factor IX nucleotide sequence, including theintron sequence, has at least 80% identity to the sequence of SEQ ID NO.3. As described above, the percentage identity may be higher.

The promoter causes expression of the nucleotide sequence encoding for afunctional factor IX protein. Any appropriate promoter may be used, suchas HLP, LP1, HCR-hAAT, ApoE-hAAT, and LSP. These promoters are describedin more detail in the following references: HLP: McIntosh J. et al.,Blood 2013 Apr. 25, 121(17):3335-44; LP1: Nathwani et al., Blood. 2006Apr. 1, 107(7): 2653-2661; HCR-hAAT: Miao et al., Mol Ther. 2000; 1:522-532; ApoE-hAAT: Okuyama et al., Human Gene Therapy, 7, 637-645(1996); and LSP: Wang et al., Proc Natl Acad Sci USA. 1999 Mar. 30,96(7): 3906-3910. A preferred promoter is also described in WO2011/005968. Preferably, the promoter is a liver specific promoter.

The promoter preferably has a nucleotide sequence which has at least 80%identity to the sequence of SEQ ID NO. 4. In some embodiments, thenucleotide sequence has at least 82% identity to the sequence of SEQ IDNO. 4. In other embodiments, the nucleotide sequence has at least 84%identity to the sequence of SEQ ID NO. 4. In further embodiments, thenucleotide sequence has at least 86% identity to the sequence of SEQ IDNO. 4. In particular embodiments, the nucleotide sequence has at least88% identity to the sequence of SEQ ID NO. 4. In some embodiments, thenucleotide sequence has at least 90% identity to the sequence of SEQ IDNO. 4. In other embodiments, the nucleotide sequence has at least 91%identity to the sequence of SEQ ID NO. 4. In further embodiments, thenucleotide sequence has at least 92% identity to the sequence of SEQ IDNO. 4. In particular embodiments, the nucleotide sequence has at least93% identity to the sequence of SEQ ID NO. 4. In some embodiments, thenucleotide sequence has at least 94% identity to the sequence of SEQ IDNO. 4. In other embodiments, the nucleotide sequence has at least 95%identity to the sequence of SEQ ID NO. 4. In further embodiments, thenucleotide sequence has at least 96% identity to the sequence of SEQ IDNO. 4. In particular embodiments, the nucleotide sequence has at least97% identity to the sequence of SEQ ID NO. 4. In some embodiments, thenucleotide sequence has at least 98% identity to the sequence of SEQ IDNO. 4. In other embodiments, the nucleotide sequence has at least 99%identity to the sequence of SEQ ID NO. 4. In preferred embodiments, thenucleotide sequence of the promoter is the sequence of SEQ ID NO. 4.

The promoter having SEQ ID NO. 4 is a liver specific promoter which hasbeen found to give particularly good expression in the liver. Whilstgiving good expression, this promoter is also relatively small whichallows more efficient packaging of the vector.

The nucleotide sequence of the promoter is preferably between 300 and400 nucleotides in length. In some embodiments, the nucleotide sequenceof the promoter is between 330 and 380 nucleotides in length. In otherembodiments, the nucleotide sequence of the promoter is between 345 and365 nucleotides in length. In particular embodiments, the nucleotidesequence of the promoter is between 350 and 360 nucleotides in length.

The vector may be any appropriate vector for expressing the FIX protein,including viral and non-viral vectors. Viral vectors include aparvovirus, an adenovirus, a retrovirus, a lentivirus or a herpessimplex virus. The parvovirus may be an adenovirus-associated virus(AAV). The vector is preferably a recombinant adeno-associated viral(rAAV) vector or a lentiviral vector. More preferably, the vector is anrAAV vector.

A vector according to the invention may be a gene delivery vector. Sucha gene delivery vector may be a viral gene delivery vector or anon-viral gene delivery vector.

Accordingly, the present invention provides gene delivery vectors basedon animal parvoviruses, in particular dependoviruses such as infectioushuman or simian AAV, and the components thereof (e.g., an animalparvovirus genome) for use as vectors for introduction and/or expressionof a factor IX protein in a mammalian cell. The term “parvoviral” asused herein thus encompasses dependoviruses such as any type of AAV.

Viruses of the Parvoviridae family are small DNA animal viruses. Thefamily Parvoviridae may be divided between two subfamilies: theParvovirinae, which infect vertebrates, and the Densovirinae, whichinfect insects. Members of the subfamily Parvovirinae are hereinreferred to as the parvoviruses and include the genus Dependovirus. Asmay be deduced from the name of their genus, members of the Dependovirusare unique in that they usually require coinfection with a helper virussuch as adenovirus or herpes virus for productive infection in cellculture. The genus Dependovirus includes AAV, which normally infectshumans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6) or primates (e.g.,serotypes 1 and 4), and related viruses that infect other warm-bloodedanimals (e.g., bovine, canine, equine, and ovine adeno-associatedviruses). Further information on parvoviruses and other members of theParvoviridae is described in Kenneth I. Berns, “Parvoviridae: TheViruses and Their Replication,” Chapter 69 in Fields Virology (3d Ed.1996). For convenience the present invention is further exemplified anddescribed herein by reference to AAV. It is, however, understood thatthe invention is not limited to AAV but may equally be applied to otherparvoviruses.

The genomic organization of all known AAV serotypes is very similar. Thegenome of AAV is a linear, single-stranded DNA molecule that is lessthan about 5,000 nucleotides (nt) in length. Inverted terminal repeats(ITRs) flank the unique coding nucleotide sequences for thenon-structural replication (Rep) proteins and the structural (VP)proteins. The VP proteins (VP1, -2 and -3) form the capsid. The terminal145 nt are self-complementary and are organized so that an energeticallystable intramolecular duplex forming a T-shaped hairpin may be formed.These hairpin structures function as an origin for viral DNAreplication, serving as primers for the cellular DNA polymerase complex.Following wild type (wt) AAV infection in mammalian cells the Rep genes(i.e. encoding Rep78 and Rep52 proteins) are expressed from the 1³5promoter and the P19 promoter, respectively, and both Rep proteins havea function in the replication of the viral genome. A splicing event inthe Rep ORF results in the expression of actually four Rep proteins(i.e. Rep78, Rep68, Rep52 and Rep40). However, it has been shown thatthe unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammaliancells are sufficient for AAV vector production. Also in insect cells theRep78 and Rep52 proteins suffice for AAV vector production.

In an AAV suitable for use as a gene therapy vector, the vector genometypically comprises a nucleic acid to be packaged for delivery to atarget cell. According to this particular embodiment, the heterologousnucleotide sequence is located between the viral ITRs at either end ofthe vector genome. In further preferred embodiments, the parvovirus(e.g. AAV) cap genes and parvovirus (e.g. AAV) rep genes are deletedfrom the template genome (and thus from the virion DNA producedtherefrom). This configuration maximizes the size of the nucleic acidsequence(s) that can be carried by the parvovirus capsid.

According to this particular embodiment, the nucleic acid is locatedbetween the viral ITRs at either end of the substrate. It is possiblefor a parvoviral genome to function with only one ITR. Thus, in a genetherapy vector of the invention based on a parvovirus, the vector genomeis flanked by at least one ITR, but, more typically, by two AAV ITRs(generally with one either side of the vector genome, i.e. one at the 5′end and one at the 3′ end). There may be intervening sequences betweenthe nucleic acid in the vector genome and one or more of the ITRs.

Preferably, the nucleotide sequence encoding a functional factor IXprotein (for expression in the mammalian cell) will be incorporated intoa parvoviral genome located between two regular ITRs or located oneither side of an ITR engineered with two D regions.

AAV sequences that may be used in the present invention for theproduction of AAV gene therapy vectors can be derived from the genome ofany AAV serotype. Generally, the AAV serotypes have genomic sequences ofsignificant homology at the amino acid and the nucleic acid levels,provide an identical set of genetic functions, produce virions which areessentially physically and functionally equivalent, and replicate andassemble by practically identical mechanisms. For the genomic sequenceof the various AAV serotypes and an overview of the genomic similaritiessee e.g. GenBank Accession number U89790; GenBank Accession numberJ01901; GenBank Accession number AF043303; GenBank Accession numberAF085716; Chiorini et al, 1997; Srivastava et al, 1983; Chiorini et al,1999; Rutledge et al, 1998; and Wu et al, 2000. AAV serotype 1, 2, 3, 4,5, 6, 7, 8 or 9 may be used in the present invention. However, AAVserotypes 1, 5 or 8 are preferred sources of AAV sequences for use inthe context of the present invention. The sequences from the AAVserotypes may be mutated or engineered when being used in the productionof gene therapy vectors.

Preferably, the AAV ITR sequences for use in the context of the presentinvention are derived from AAV1, AAV2, AAV4 and/or AAV6. Likewise, theRep (Rep78 and Rep52) coding sequences are preferably derived from AAV1,AAV2, AAV4 and/or AAV6. The sequences coding for the VP1, VP2, and VP3capsid proteins for use in the context of the present invention mayhowever be taken from any of the known 42 serotypes, more preferablyfrom AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 or newlydeveloped AAV-like particles obtained by e.g. capsid shufflingtechniques and AAV capsid libraries.

AAV Rep and ITR sequences are particularly conserved among mostserotypes. The Rep78 proteins of various AAV serotypes are e.g. morethan 89% identical and the total nucleotide sequence identity at thegenome level between AAV2, AAV3A, AAV3B, and AAV6 is around 82%(Bantel-Schaal et al, 1999). Moreover, the Rep sequences and ITRs ofmany AAV serotypes are known to efficiently cross-complement (i.e.,functionally substitute) corresponding sequences from other serotypes inproduction of AAV particles in mammalian cells. US 2003148506 reportsthat AAV Rep and ITR sequences also efficiently cross-complement otherAAV Rep and ITR sequences in insect cells.

The AAV VP proteins are known to determine the cellular tropicity of theAAV virion. The VP protein-encoding sequences are significantly lessconserved than Rep proteins and genes among different AAV serotypes. Theability of Rep and ITR sequences to cross-complement correspondingsequences of other serotypes allows for the production of pseudotypedAAV particles comprising the capsid proteins of a serotype (e.g., AAV1,5 or 8) and the Rep and/or ITR sequences of another AAV serotype (e.g.,AAV2). Such pseudotyped rAAV particles are a part of the presentinvention.

Modified “AAV” sequences also can be used in the context of the presentinvention, e.g. for the production of AAV gene therapy vectors. Suchmodified sequences e.g. include sequences having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or more nucleotide and/or amino acid sequenceidentity (e.g., a sequence having about 75-99% nucleotide sequenceidentity) to an AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8 or AAVSITR, Rep, or VP can be used in place of wild-type AAV ITR, Rep, or VPsequences.

Although similar to other AAV serotypes in many respects, AAVS differsfrom other human and simian AAV serotypes more than other known humanand simian serotypes. In view thereof, the production of rAAV5 candiffer from production of other serotypes in insect cells. Where methodsof the invention are employed to produce rAAV5, it is preferred that oneor more constructs comprising, collectively in the case of more than oneconstruct, a nucleotide sequence comprising an AAVS ITR, a nucleotidesequence comprises an AAVS Rep coding sequence (i.e. a nucleotidesequence comprises an AAVS Rep78). Such ITR and Rep sequences can bemodified as desired to obtain efficient production of AAVS orpseudotyped AAVS vectors. For example, the start codon of the Repsequences can be modified, VP splice sites can be modified oreliminated, and/or the VP1 start codon and nearby nucleotides can bemodified to improve the production of AAV5 vectors.

Thus, the viral capsid used in the invention may be from any parvovirus,either an autonomous parvovirus or dependovirus, as described above.Preferably, the viral capsid is an AAV capsid (e. g., AAV1, AAV2, AAV3,AAV4, AAV5 or AAV6 capsid). In general, the AAV1 capsid or AAV6 capsidare preferred. The choice of parvovirus capsid may be based on a numberof considerations as known in the art, e.g., the target cell type, thedesired level of expression, the nature of the heterologous nucleotidesequence to be expressed, issues related to viral production, and thelike. For example, the AAV1 and AAV6 capsid may be advantageouslyemployed for skeletal muscle; AAV1, AAV5 and AAV8 for the liver andcells of the central nervous system (e.g., brain); AAV5 for cells in theairway and lung or brain; AAV3 for bone marrow cells; and AAV4 forparticular cells in the brain (e. g., appendable cells).

It is within the technical skills of the skilled person to select themost appropriate virus, virus subtype or virus serotype. Some subtypesor serotypes may be more appropriate than others for a certain type oftissue.

For example, liver-specific expression of a nucleic acid of theinvention may advantageously be induced by AAV-mediated transduction ofliver cells. Liver is amenable to AAV-mediated transduction, anddifferent serotypes may be used (for example, AAV1, AAV5 or AAV8).Transduction of muscle may be accomplished by administration of an AAVencoding a nucleic acid via the blood stream. Thus, intravenous orintra-arterial administration is applicable.

A parvovirus gene therapy vector prepared according to the invention maybe a “hybrid” particle in which the viral ITRs and viral capsid are fromdifferent parvoviruses. Preferably, the viral TRs and capsid are fromdifferent serotypes of AAV. Likewise, the parvovirus may have a“chimeric” capsid (e. g., containing sequences from differentparvoviruses, preferably different AAV serotypes) or a “targeted” capsid(e. g., a directed tropism).

In the context of the invention “at least one parvoviral ITR nucleotidesequence” is understood to mean a palindromic sequence, comprisingmostly complementary, symmetrically arranged sequences also referred toas “A,” “B,” and “C” regions. The ITR functions as an origin ofreplication, a site having a “cis” role in replication, i.e., being arecognition site for trans-acting replication proteins such as e.g. Rep78 (or Rep68) which recognize the palindrome and specific sequencesinternal to the palindrome. One exception to the symmetry of the ITRsequence is the “D” region of the ITR. It is unique (not having acomplement within one ITR). Nicking of single-stranded DNA occurs at thejunction between the A and D regions. It is the region where new DNAsynthesis initiates. The D region normally sits to one side of thepalindrome and provides directionality to the nucleic acid replicationstep. A parvovirus replicating in a mammalian cell typically has two ITRsequences. It is, however, possible to engineer an ITR so that bindingsites are on both strands of the A regions and D regions are locatedsymmetrically, one on each side of the palindrome. On a double-strandedcircular DNA template (e.g., a plasmid), the Rep78- or Rep68-assistednucleic acid replication then proceeds in both directions and a singleITR suffices for parvoviral replication of a circular vector. Thus, oneITR nucleotide sequence can be used in the context of the presentinvention. Preferably, however, two or another even number of regularITRs are used. Most preferably, two ITR sequences are used. A preferredparvoviral ITR is an AAV ITR. For safety reasons it may be desirable toconstruct a parvoviral (AAV) vector that is unable to further propagateafter initial introduction into a cell. Such a safety mechanism forlimiting undesirable vector propagation in a recipient may be providedby using AAV with a chimeric ITR as described in US 2003148506.

Those skilled in the art will appreciate that the viral Rep protein(s)used for producing an AAV vector of the invention may be selected withconsideration for the source of the viral ITRs. For example, the AAVSITR typically interacts more efficiently with the AAVS Rep protein,although it is not necessary that the serotype of ITR and Rep protein(s)are matched.

The ITR(s) used in the invention are typically functional, i.e. they maybe fully resolvable and are preferably AAV sequences, with serotypes 1,2, 3, 4, 5 or 6 being preferred. Resolvable AAV ITRs according to thepresent invention need not have a wild-type ITR sequence (e. g., awild-type sequence may be altered by insertion, deletion, truncation ormissense mutations), as long as the ITR mediates the desired functions,e. g., virus packaging, integration, and/or provirus rescue, and thelike.

Advantageously, by using a gene therapy vector as compared with previousapproaches, the restoration of protein synthesis, i.e. factor IXsynthesis, is a characteristic that the transduced cells acquirepermanently or for a sustained period of time, thus avoiding the needfor continuous administration to achieve a therapeutic effect.

Accordingly, the vectors of the invention therefore represent a tool forthe development of strategies for the in vivo delivery of a FIXnucleotide sequence, by engineering the nucleic acid within a genetherapy vector that efficiently transduces an appropriate cell type,such as a liver cell.

Preferably, the vector is a single stranded vector rather than aself-complementary vector. Surprisingly, this has been shown to givebetter protein expression.

The vector may further comprise a poly A tail. Preferably, this ispositioned downstream of the nucleotide sequence encoding for afunctional FIX protein. Preferably, the poly A tail is a bovine growthhormone poly A tail (bGHpA). Preferably, this is between 250 and 270nucleotides in length.

In a preferred embodiment, the vector comprises a nucleotide sequencewhich has 80% identity to the sequence of SEQ ID NO. 5. In someembodiments, the nucleotide sequence has at least 82% identity to thesequence of SEQ ID NO. 5. In other embodiments, the nucleotide sequencehas at least 84% identity to the sequence of SEQ ID NO. 5. In furtherembodiments, the nucleotide sequence has at least 86% identity to thesequence of SEQ ID NO. 5. In particular embodiments, the nucleotidesequence has at least 88% identity to the sequence of SEQ ID NO. 5. Insome embodiments, the nucleotide sequence has at least 90% identity tothe sequence of SEQ ID NO. 5. In other embodiments, the nucleotidesequence has at least 91% identity to the sequence of SEQ ID NO. 5. Infurther embodiments, the nucleotide sequence has at least 92% identityto the sequence of SEQ ID NO. 5. In particular embodiments, thenucleotide sequence has at least 93% identity to the sequence of SEQ IDNO. 5. In some embodiments, the nucleotide sequence has at least 94%identity to the sequence of SEQ ID NO. 5. In other embodiments, thenucleotide sequence has at least 95% identity to the sequence of SEQ IDNO. 5. In further embodiments, the nucleotide sequence has at least 96%identity to the sequence of SEQ ID NO. 5. In particular embodiments, thenucleotide sequence has at least 97% identity to the sequence of SEQ IDNO. 5. In some embodiments, the nucleotide sequence has at least 98%identity to the sequence of SEQ ID NO. 5. In other embodiments, thenucleotide sequence has at least 99% identity to the sequence of SEQ IDNO. 5. In preferred embodiments, the vector comprises the nucleotidesequence of SEQ ID NO. 5.

In another aspect of the invention, there is provided a nucleic acidmolecule comprising a nucleotide sequence encoding for a functionalfactor IX protein, wherein exons 3 to 5 of the nucleotide sequence haveat least 80% identity to the sequence of SEQ ID NO. 6.

The invention also provides a vector comprising a nucleotide sequenceencoding for a functional factor IX protein, wherein exons 3 to 5 of thenucleotide sequence have at least 80% identity to the sequence of SEQ IDNO. 6. The vector will comprise other elements to allow the functionalFIX protein to be expressed such as a promoter. Such elements are wellknown to a person skilled in the art.

Additional features relating to the nucleotide sequence encoding for afunctional factor IX protein, and exons 3 to 5 of the sequence, aredescribed above.

In another aspect of the invention, there is provided a nucleic acidmolecule comprising a nucleotide sequence encoding for a functionalfactor IX protein, the nucleotide sequence having at least 80% identityto the sequence of SEQ ID NO. 2.

The invention also provides a vector comprising a nucleotide sequenceencoding for a functional factor IX protein, the nucleotide sequencehaving at least 80% identity to the sequence of SEQ ID NO. 2. The vectorwill comprise other elements to allow the functional FIX protein to beexpressed such as a promoter. Such elements are well known to a personskilled in the art.

Additional features relating to the nucleotide sequence encoding for afunctional factor IX protein are described above.

In another aspect of the invention, there is provided a nucleic acidmolecule comprising a nucleotide sequence encoding for a functionalfactor IX protein and containing an intron sequence positioned betweenexon 1 and exon 2 of the factor IX sequence, wherein the nucleotidesequence has at least 80% identity to the sequence of SEQ ID NO. 3.

Additional features relating to the nucleotide sequence encoding for afunctional factor IX protein and which contains an intron sequence aredescribed above.

In another aspect of the invention, there is provided a nucleic acidmolecule comprising an intron sequence having at least 80% identity tothe sequence of SEQ ID NO. 1.

Additional features relating to the intron sequence are described above.

In another aspect of the invention, there is provided a nucleic acidmolecule comprising a promoter, wherein the promoter has a nucleotidesequence having at least 80% identity to the sequence of SEQ ID NO. 4.

Additional features relating to the promoter sequence are describedabove.

Preferably, the nucleic acids described above are isolated.

It would be well with the capabilities of a skilled person to producethe nucleic acid molecules described above. This could be done, forexample, using chemical synthesis of a given sequence.

Further, a skilled person would readily be able to determine whether anucleic acid expresses a functional protein. Suitable methods would beapparent to those skilled in the art. For example, one suitable in vitromethod involves inserting the nucleic acid into a vector, such as alentiviral or an AAV vector, transducing host cells, such as 293T orHeLa cells, with the vector, and assaying for factor IX activity.Alternatively, a suitable in vivo method involves transducing a vectorcontaining the nucleic acid into haemophiliac mice and assaying forfunctional factor IX in the plasma of the mice. Suitable methods aredescribed in more detail below.

The nucleic acid can be any type of nucleic acid composed ofnucleotides. The nucleic acid should be able to be expressed so that aprotein is produced. Preferably, the nucleic acid is DNA or RNA.

The invention also provides a host cell comprising any one of thenucleic acid molecules or vectors described above. Preferably, thevector is capable of expressing the FIX nucleotide sequence in the host.The host may be any suitable host.

As used herein, the term “host” refers to organisms and/or cells whichharbour a nucleic acid molecule or a vector of the invention, as well asorganisms and/or cells that are suitable for use in expressing arecombinant gene or protein. It is not intended that the presentinvention be limited to any particular type of cell or organism. Indeed,it is contemplated that any suitable organism and/or cell will find usein the present invention as a host. A host cell may be in the form of asingle cell, a population of similar or different cells, for example inthe form of a culture (such as a liquid culture or a culture on a solidsubstrate), an organism or part thereof

A host cell according to the invention may permit the expression of anucleic acid molecule of the invention. Thus, the host cell may be, forexample, a bacterial, a yeast, an insect or a mammalian cell.

In addition, the invention provides a transgenic animal comprising cellscomprising the nucleic acid molecule encoding for a functional FIXprotein described above or a vector described above. Preferably theanimal is a non-human mammal, especially a primate. Alternatively, theanimal may be a rodent, especially a mouse; or may be canine, feline,ovine or porcine.

In one aspect, the invention provides a pharmaceutical compositioncomprising a nucleic acid molecule or a vector of the invention and oneor more pharmaceutically acceptable excipients. The one or moreexcipients include carriers, diluents and/or other medicinal agents,pharmaceutical agents or adjuvants, etc.

The invention also provides a method of treating haemophilia Bcomprising administering a therapeutically effective amount of a vectoras described above to a patient suffering from haemophilia B.Preferably, the patient is human.

When haemophilia B is “treated” in the above method, this means that oneor more symptoms of haemophilia are ameliorated. It does not mean thatthe symptoms of haemophilia are completely remedied so that they are nolonger present in the patient, although in some methods, this may be thecase. The method of treating results in one or more of the symptoms ofhaemophilia B being less severe than before treatment.

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, such as raising the level of functional factor IX ina subject (so as to lead to functional factor IX production to a levelsufficient to ameliorate the symptoms of haemophilia B).

Delivery of a nucleic acid or vector of the invention to a host cell invivo may result in an increase of functional factor IX in the host, forexample to a level that ameliorates one or more symptoms of haemophiliaB.

The level of naturally occurring factor IX in a subject suffering fromhaemophilia B varies depending on the severity of the haemophilia.Patients with a severe form of the disease have factor IX levels of lessthan about 1% of the level found in a normal healthy subject (referredto herein as “a normal level”). It has been found that when the methodof treatment of the invention is used, it can cause an increase in thelevel of functional factor IX to at least about 1% of normal levels. Ina subject suffering from haemophilia B, an increase in circulating FIXto >1% of normal levels can significantly ameliorate the severe bleedingphenotype. In some embodiments, the method of treatment of the inventioncauses an increase in the level of functional factor IX to at leastabout 2%, at least about 3%, at least about 4%, at least about 10%, atleast about 15%, at least about 20% or at least about 25% of normallevels. In a particular embodiment, the method of treatment of theinvention causes an increase in the level of functional factor IX to atleast about 30% of normal levels. This level of increase would virtuallynormalise coagulation of blood in subjects suffering haemophilia B. Suchsubjects are unlikely to require factor IX concentrates following traumaor during surgery.

In one embodiment, the method of treatment of the invention causes anincrease in the level of functional factor IX to, at most, normallevels.

The level of functional factor IX can be measured relatively easily andmethods for measuring factor IX levels are well known to those skilledin the art. Many clotting assays are available, including chromogenicand clotting based assays. ELISA tests are also widely available.

Further, the invention provides the nucleic acid molecule encoding for afunctional FIX protein as described above or a vector as described abovefor use in therapy, for example, in the treatment of haemophilia B.

In addition, the invention provides the use of the nucleic acid moleculeencoding for a functional FIX protein as described above or a vector asdescribed above in the manufacture of a medicament for treatinghaemophilia B.

The invention also provided a method for delivery of a nucleotidesequence encoding a functional FIX protein to a subject, which methodcomprises administering to the said subject a nucleic acid moleculeencoding a functional FIX protein as described above or a vector asdescribed above.

In the description above, the term “identity” is used to refer to thesimilarity of two sequences. For the purpose of this invention, it isdefined here that in order to determine the percent identity of twonucleotide sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a firstnucleic acid for optimal alignment with a second amino or nucleic acidsequence). The nucleotide residues at nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid or nucleotide residue as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical positions/total number of positions (i.e. overlappingpositions)×100). Preferably, the two sequences are the same length. Asequence comparison is typically carried out over the entire length ofthe two sequences being compared.

The skilled person will be aware of the fact that several differentcomputer programs are available to determine the homology or identitybetween two sequences. For instance, a comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using a mathematical algorithm. In a preferred embodiment,the percent identity between two nucleic acid sequences is determinedusing the sequence alignment software Clone Manager 9 (Sci-Edsoftware—www.scied.com) using global DNA alignment; parameters: bothstrands; scoring matrix: linear (mismatch 2, OpenGap 4, ExtGap 1).

Alternatively, the percent identity between two amino acid or nucleicacid sequences is determined using the Needleman and Wunsch (1970)algorithm which has been incorporated into the GAP program in theAccelrys GCG software package (available athttp://www.accelrys.com/products/gcg/), using either a Blosum 62 matrixor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid sequences of the present invention can further be usedas a “query sequence” to perform a search against public databases to,for example, identify other family members or related sequences. Suchsearches can be performed using the BLASTN programs (version 2.0) ofAltschul, et al, 1990. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al,1997. When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (e.g. BLASTN) can be used. See thehomepage of the National Center for Biotechnology Information at hill)://www.ncbi.nlm.nih.gov/.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

A skilled person will appreciate that all aspects of the invention,whether they relate to, for example, the nucleic acid, the vector, thehost cell or the use, are equally applicable to all other aspects of theinvention. In particular, aspects of the method of treatment, forexample, the administration of the nucleic acid or vector, may have beendescribed in greater detail than in some of the other aspects of theinvention, for example, relating to the use of the nucleic acid orvector for treating haemophilia B. However, the skilled person willappreciate where more detailed information has been given for aparticular aspect of the invention, this information is generallyequally applicable to other aspects of the invention. Further, theskilled person will also appreciate that the description relating to themethod of treatment is equally applicable to the use of the nucleic acidor vector in treating haemophilia B.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1 shows two vector constructs for delivering factor IX. The topconstruct HCR hAAT FIX is an existing factor IX gene expression vectorused in a gene therapy trial. The bottom construct HCR hAAT FIX TI isthe same except that it uses a truncated intron.

FIG. 2 shows FIX expression of the two constructs shown in FIG. 1 inmice following tail vein administration of an identical dose of vector.Expression levels for the HCR hAAT TI FIX vector were 1.8 fold higherthan for the HCR hAAT FIX vector which was unexpected based on the priorart.

FIG. 3 shows two factor IX vector constructs. The top constructHCR-hAAT-FIX comprises the wild type factor IX sequence whereas thebottom construct HCR-hAAT-codop-FIX comprises a codon optimised factorIX sequence. In this sequence, exons 1 and 2 have the wild typesequence, whilst exons 3 to 5 have the codon optimised sequence.

FIG. 4 shows FIX expression of the two constructs shown in FIG. 3 inmice following tail vein administration at two doses of vector.Expression levels for the HCR-hAAT-codop-FIX are significantly higherthan for the HCR-hAAT-FIX vector.

FIG. 5 shows two further vector constructs for delivering factor IX. Thetop construct scAAV-LP1-FIXco is a self-complementary vector being usedin a haemophilia B clinical trial. The bottom constructscAAV-HLP2-TI-codop-FIX is a single stranded vector which uses a newliver specific promoter (HLP2). In this construct, exons 1 and 2 havethe wild type sequence, whilst exons 3 to 5 have the codon optimisedsequence.

FIG. 6 shows FIX expression of the two constructs shown in FIG. 5 inmice following tail vein administration of an equivalent number ofvector particles as assessed by a gel based titration method. AAV8capsid pseudotyped single stranded HLP2-TI-codop-FIX mediated at least 3fold higher levels of FIX when compared to scAAV-LP1-FIXco. This issurprising as self-complementary vectors have previously been shown tomediate substantially higher levels of expression than possible withsingle-stranded AAV-FIX constructs (Wu et al. Mol Ther. 2008 February;16(2):280-9 and Nathwani et al. Blood. 2006 Apr. 1; 107(7):2653-61).However, these data show that when optimally configured with regards toinclusion of a strong promoter and efficient splice sites, a singlestranded AAV can mediate higher levels of transgene expression thanachievable with self-complementary AAV.

DETAILED DESCRIPTION

The overriding goal of the inventors' research program is to establish acure for haemophilia B (HB) that is safe, effective and widelyavailable. They established proof-of-concept in a pivotal clinical trialin which a single peripheral vein administration of a self-complementary(sc) adeno-associated viral vector (AAV) expressing a codon optimisedFIX transgene (scAAV2/8-LP1-hFIXco) resulted in: (1) stable (>48 months)expression of FIX at 16% without long lasting toxicity; (2)discontinuation of prophylaxis in 4/7 participants; (3) reduction inannual bleeding rate of >90% for the 6 subjects in the high dose cohort;and (4) a cost saving so far of £1.5M from reduction in FIX concentrateusage (Nathwani A C et al. N Engl J Med. 365:2357-65, 2011). Obstaclesremain to the overriding goal of making AAV-mediated transfer of thenormal FIX gene the world-wide curative standard-of-care. Foremost isthe body's immune response to cells that have been transduced with theviral vector, resulting in asymptomatic, transient elevation of serumliver enzymes, suggesting local inflammation in the liver. This adverseevent only occurred at the high dose but was relatively common (n=4/6).The inventors' efforts have therefore focused on improving potency andtransduction efficiency of AAV vectors to enable therapeutic genetransfer in humans with lower, potentially safer vector doses. Inpursuit of this goal, the inventors have developed a new more potent FIXexpression cassette called HLP2-TI-codop-FIX for AAV mediated genetherapy of haemophilia B.

An initial evaluation compared a single stranded HCR hAAT FIX constructcontaining a truncated intron 1 (HCR-hAAT-TI-FIX) to an identicalconstruct (HCR-hAAT-FIX) currently being used in an on-going genetherapy trial in mice following tail vein administration of an identicaldose of vector. In brief, a dose of lel 1 vg was administered into thetail vein of 4-6 week old male C57B1/6 mice (N=4-6 animals/group). Thevector dose was assessed by a gel based titration method describedpreviously (Fagone et al., Hum Gene Ther Methods. 2012 Feb. 23 (1):1-7).FIX levels were assessed using the previously described ELISA method at4 weeks after gene transfer (Nathwani et al., Mol Ther. 2011 May 19.(5):876-85). A 1.8 fold higher level of FIX in the cohort transducedwith HCR hAAT TI FIX was observed per copy of the AAV-FIX transgene (asassessed by a PCR quantification method using primers to hAAT) in theliver at 4 weeks, which was unexpected based on prior art (FIG. 1).

The DNA sequences in HCR-hAAT-FIX were further modified using ourin-house codon-optimization algorithm in which codons in the FIX cDNAfor a given amino-acid were substituted with the codon most frequentlyused by the human albumin gene for the same amino-acid since the humanalbumin is expressed in abundance by the liver. The resulting codop-FIXcDNA was 85% identical to that previously used by our groupscAAV-LP1-FIXco (Nathwani et al., Blood. 2006 Apr. 1. 107(7):2653-61).The codop-FIX cDNA was synthesized and cloned downstream of the HCR-hAATpromoter (FIG. 3). To assess the potency of HCR-hAAT-codop-FIX, serotype8 pseudotyped vector was injected into 4-6 week old male C57B1/6 mice ata dose of 2e9 or 2e10vg/mouse (N=4-6 animals/dose) based on a gel basedtitration method. FIX expression in murine plasma was assessed by ELISAat 4 weeks after gene transfer in each dose cohort and compared with thelevels achieved in identical dose cohorts transduced with HCR-hAAT-FIX,a vector that contains wild type nucleotide sequence in the FIX cDNA.Codon optimisation of the FIX cDNA resulted in a statistically (onesample t test) significant improvement in transgene gene expression atboth dose levels as illustrated in FIG. 4.

Next, the inventors compared the potency of single strandedHLP2-TI-codop-FIX with a self-complementary LP1-FIXco expressioncassette currently being used in a haemophilia B clinical trial. Inbrief, both vectors pseudotyped with serotype 8 capsid were titeredusing the gel based method to ensure equivalent numbers of selfcomplementary and single stranded AAV particles were administered in 4-8week old male C57B1/6 mice. Although transduction with single strandedAAV vectors is limited by the need to convert the single-stranded genometo transcriptionally active double-stranded forms, a head to headcomparison showed that for a given vector dose HLP2-TI-codop-FIXmediated at least 3 fold higher levels of FIX in plasma of mice for agiven copy of vector in the liver when compared to scAAV-LP1-FIXco (FIG.6) at 4 weeks after gene transfer, despite the fact thatself-complementary vectors are more efficient at forming double strandedtranscriptionally active units in the liver.

SEQUENCES

SEQ ID NO. 1—Nucleotide sequence of truncated intron (TI).

SEQ ID NO. 2—Nucleotide sequence of codon optimised FIX. Features: FIXExon 1: 1-88; FIX Exons 2-5: 89-1386.

SEQ ID NO. 3—Nucleotide sequence of codon optimised FIX containingtruncated intron (TI). Features: FIX Exon 1: 1-88; Truncated intron:89-387; FIX Exon 2-5: 388-1685.

SEQ ID NO. 4—Nucleotide sequence of promoter HLP2.

SEQ ID NO. 5—Nucleotide sequence of HLP2 FIX TI vector. Features: HLP2:1-354; FIX Exon 1: 425-512; Truncated intron (TI): 513-811; FIX Exons2-5: 812-2109; bGHpA: 2125-2383.

SEQ ID NO. 6—Nucleotide sequence of codon optimised exons 3 to 5 of FIX.

1.-46. (canceled)
 47. A vector for expressing factor IX protein, thevector comprising a promoter, a nucleotide sequence encoding for afunctional factor IX protein, and an intron sequence, wherein the intronsequence is positioned between exon 1 and exon 2 of the nucleotidesequence encoding for the functional factor IX protein, and wherein: theintron sequence has at least 80% identity to the sequence of SEQ ID NO.1, the intron sequence has at least 95% identity to the sequence of SEQID NO. 1, or the intron sequence has the sequence of SEQ ID NO.
 1. 48.The vector of claim 47, wherein: the promoter has a nucleotide sequencewhich has at least 80% identity to the sequence of SEQ ID NO. 4, thepromoter has a nucleotide sequence which has at least 95% identity tothe sequence of SEQ ID NO. 4, or the promoter has the nucleotidesequence of SEQ ID NO.
 4. 49. The vector of claim 47, wherein: thenucleotide sequence encoding for the functional protein has at least 80%identity to the sequence of SEQ ID NO. 2, the nucleotide sequenceencoding for the functional FIX protein has at least 95% identity to thesequence of SEQ ID NO. 2, or the nucleotide sequence encoding for thefunctional FIX protein has the sequence of SEQ ID NO.
 2. 50. The vectorof claim 47, wherein: the nucleotide sequence encoding the functionalFIX protein has at least 80% identity to the sequence of SEQ ID NO. 6,the nucleotide sequence encoding the functional FIX protein has at least95% identity to the sequence of SEQ ID NO. 6, or the nucleotide sequenceencoding the functional FIX protein has the sequence of SEQ ID NO. 6.51. The vector of claim 47, wherein: the nucleotide sequence encodingfor the functional FIX protein, including the intron sequence betweenexon 1 and 2, has 80% identity to the sequence of SEQ ID NO. 3, thenucleotide sequence encoding for the functional FIX protein, includingthe intron sequence between exon 1 and 2, has 95% identity to thesequence of SEQ ID NO. 3, or the nucleotide sequence encoding for thefunctional FIX protein, including the intron sequence between exon 1 and2, has the sequence of SEQ ID NO.
 3. 52. The vector of claim 47, whereinthe nucleotide sequence encoding for the functional factor IX proteincomprises: a nucleotide sequence that has 80% identity, 95% identity, or100% identity to nucleotides 1-88 (exon 1) of Genbank accession numberJ00137.1; or a nucleotide sequence that has 80% identity, 95% identity;or 100% identity to nucleotides 89-197 (exon 2, partial) of Genbankaccession number J00137.1.
 53. The vector of claim 52, wherein thenucleotide sequence encoding for the functional factor IX protein iscodon optimized.
 54. The vector of claim 53, wherein the nucleotidesequence encoding for the functional factor IX protein comprises anucleotide sequence that has 80% identity to Genbank accession numberJ00137.1
 55. The vector of claim 47; wherein: the vector comprises anucleotide sequence which has 80% identity to the sequence of SEQ ID NO.5, the vector comprises a nucleotide sequence which has 95% identity tothe sequence of SEQ ID NO. 5, or the vector comprises a nucleotidesequence which has the sequence of SEQ ID NO.
 5. 56. The vector of claim47, wherein: the intron sequence has at least 95% identity to thesequence of SEQ ID NO. 1, the nucleotide sequence encoding for thefunctional FIX protein has at least 95% identity to the sequence of SEQID NO. 2, and the promoter has a nucleotide sequence which has at least95% identity to the sequence of SEQ ID NO. 4, and wherein the vector isa single stranded vector.
 57. The vector of claim 47, wherein thevector: is an AAV vector, is a single stranded vector, or furthercomprises a bovine growth hormone poly A tail.
 58. A pharmaceuticalcomposition comprising the vector of claim 47, and one or morepharmaceutically acceptable excipients.
 59. A method of treatinghaemophilia B comprising administering a therapeutically effectiveamount of the vector of claim 47 to a patient suffering from haemophiliaB.